Obesity is associated with increased risk for the development of type 2 diabetes mellitus and cardiovascular disorders. New frontiers in therapeutic intervention against obesity lie in the recognition of novel mechanisms involved during the initial stages of weight gain in humans. There has been increasing interest in the role leptin, a peripheral circulating satiety factor, which plays a key role in balancing energy expenditure and preventing fat gain. However, leptin is found at higher levels in obese humans than in non-obese humans. The failure of elevated leptin to elicit weight loss in common forms of human obesity suggests the attenuation of leptin action (leptin resistance). To this end, we propose a novel hypothesis that resistance to leptin signaling is a result of molecular and structural changes in caveolar membrane microdomains. To test our hypothesis we propose a series of studies directed at investigating the molecular mechanisms involved in disruption of leptin signaling during weight gain, using abdominal adipose tissue obtained from healthy human subjects who gain body fat in response to an overfeeding protocol. We also propose to investigate the reversibility of these changes during fat loss. In our preliminary studies we have observed first, increases in circulating leptin levels during weight gain accompanied by decreased endothelium mediated vasodilation and increased adipocyte fat accumulation. Second, increases in caveolin-1 and endothelial nitric oxide synthase (eNOS) expression during weight gain and reversal of these changes during weight loss. Third, increases in leptin-dependent caveolin-1 expression in vitro. Fourth, the ability of caveolin-1 to inhibit leptin signaling in-vitro. These observations point to a probable role of caveolin-1 and caveolar microdomains in disruption of leptin signaling as a feedback mechanism in response to high leptin levels. In this proposal we will combine histological and molecular biologic approaches to study the morphological and compositional changes in caveolar membrane microdomains associated with weight gain and weight loss (AIM#1), and the role of caveolin-1 during weight gain and weight loss in adipocytes and adipose tissue microvasculature (AIM#2). We hypothesize that during weight gain, increased circulating leptin leads to increased caveolin-1 expression in adipose tissue, which in turn leads to increased caveolin-1 and Ob-R interaction in the caveolar microdomains. This increased interaction may result in disruption of downstream Ob-R signaling which would then lead to leptin resistance. Attenuation of leptin signaling will result in increased lipid accumulation and decreased eNOS activity. The uniqueness and translational strength of this proposal lies in studying at the molecular level dynamic weight changes in human subjects, and in our ability to differentially study them in adipocytes and adipose tissue microvessels along with in-vitro studies. The long term significance of the proposal will be in understanding the mechanisms involved in attenuation of leptin signaling during weight gain in human subjects and its reversibility during weight loss. This understanding would be pivotal in development of therapeutics related to leptin resistance and obesity. PUBLIC HEALTH RELEVANCE: The proposed studies will have clear and important implications regarding the molecular mechanisms involved in attenuation of leptin signaling during weight gain and its reversibility during weight loss. In addition the results of our proposed studies may be critical in development of therapeutics relating to leptin resistance and obesity.